Use of bacteria endowed with arginine deiminase to induce apoptosis and/or reduce an inflammatory reaction and pharmaceutical or dietetic compositions containing such bacteria

ABSTRACT

Disclosed is the use of bacteria endowed with arginine deiminase to induce apoptosis and/or reduce an inflammatory reaction, and pharmaceutical or dietetic compositions containing such bacteria. Also included is a strain of  Lactobacillus brevis  highly endowed with arginine deiminase.

The present invention concerns the use of bacteria endowed with argininedeiminase to induce apoptosis and/or reduce an inflammatory reaction,and pharmaceutical or dietetic compositions which contain such bacteria.The invention also concerns a strain of Lactobacillus brevis which ishighly endowed with arginine deiminase.

The balance between the cell population in an organism can be controlledby way of regulating the rate of proliferation or differentiation ordeath of the constituent cells (Collins, M. K. L. et al. A. TrendsBiochem. Sci. 18:307, 1993). Cell death during embryogenesis,metamorphosis, hormone-dependent tissue atrophy and the normal turnoverof the tissues is referred to as “programmed cell death”. For the largepart that event takes place by way of “apoptosis”, a process which ischaracterised by condensation and segmentation of the nucleus,condensation and fragmentation of the cytoplasm and often fragmentationof the chromosomic DNA into nucleosomal units (Schwartz. L. M. et alImmunol. Today 14:582, 1993). Apoptosis in the development ofvertebrates often occurs when the cells do not receive the extracellularsurvival signals necessary to suppress an intrinsic cell suicideprogramme; the survival factors can be produced by the surrounding cellsof different type (paracrine mechanism) or of the same type (autocrinemechanism). Apoptosis occurs during embryonic development in particularin complex organs where a given cell sub-population is killed. Forexample many neurons move in the brain during development, just asauto-reactive T lymphocytes are eliminated in the interior of thethymus. In an adult apoptosis occurs in particular in tissues which aresubjected to reversible expansion as in the hormone-dependent cells ofthe breast and the prostate gland after removal of the hormone orfollowing cytokine-dependent expansion of the haemopoietic cells of thebone marrow.

The modifications which occur in the cell in the course of apoptosishave been widely studied and described (Cohen. J. J. et al Lab. Clin.Med. 124:761, 1994). Apoptosis is clearly different from necrosis whichcorresponds to the modifications which occur when cell death derivesfrom cell damage. In necrosis in fact the damaged cells swell up andburst, releasing their intracellular content which is toxic in relationto other cells of the tissue, and triggering off an inflammatoryresponse. In contrast phagocytosis of the apoptotic bodies is so fast asnot to induce dispersion of the cellular contents in the extracellularspace which otherwise would cause perilesional phlogosis typical ofnecrosis.

Recent experimental evidence indicates that alterations in cell survivalcontribute to the pathogenesis of many human diseases including cancer,viral infections, auto-immune diseases, neurovegetative disorders andAIDS (Thompson, C. B. Science 267:1456, 1995). A treatment aimed atspecifically altering apoptosis can have the potential to modify thenatural progression of some of those diseases. Both chemotherapy agentsand radiation induce the death of tumour cells primarily causing damageto the DNA which in turn causes cell suicide. In addition many tumoursconserve some of the physiological cell death control systems which arecharacteristic of the cells from which they originate. For examplecancer of the prostate and cancer of the breast are respectivelyandrogen- and oestrogen-dependent. Therefore anti-androgenic therapy inthe treatment of cancer of the prostate gland or removal of oestrogensby means of anti-oestrogens such as tamoxifen in the course of breastcancer are fundamental and universally accepted procedures. Both thosemethods induce apoptosis in the tumour cells which are otherwiserespectively dependent for their survival on androgens or oestrogens. Inaddition the beneficial effects of glucocorticoids observed in subjectswith lymphoidal leukaemia can be attributed to the induction ofapoptosis: other substances used for chemotherapy of cancer such ascyclophosphamide, metotrexate, etoposide and cisplatin induce apoptosisof tumour cells (Thatte. U. et al Apoptosis, Drugs 54:511, 1997).

Previous studies have shown that lactic bacteria present in foods and/orin dietetic/pharmaceutical formulations can cause transitorycolonisation of the intestine and have beneficial effects. Survivalduring the intestinal transit or adhesion to the epithelium seem to beis important for modifying the immune response of the host (Schiffrin.E. J. et al Am. J. Clin. Nutr. 66: 515S, 1997). The potentiallybeneficial effects of lactic bacteria include protection from entericinfections, stimulating the secretion of IgA, and inhibition of thegrowth of intestinal carcinoma, strengthening the activity of IgA.T-cells and macrophages (Perdigon. G. et al J. Dairy Sci. 78:1597,1995). In vitro, lactic bacteria have revealed a capacity to stimulatethe production of alpha TNF, interleukin (IL)-6 and IL-10 on the part ofhuman mononuclear cells, even to an extent greater than that revealedwhen using lipopolysaccharide (LPS) as a stimulating agent, confirming apotentiating action on non-specific immunity of the host (Miettinen, M.et al Infect. Immun. 64:5403, 1996). Still in vitro, lactic bacteriahave demonstrated a capacity to absorb mutagenic substances present incooked foods, confirming the observation that the administration oflactobacilli in man reduces the excretion of mutagenic substance afterthe ingestion of fried meat and thus the risk of cancer of the colon(Lidbeck, A. et al Eur. J. Cancer Prev. 1:341, 1992). Experimentsconducted with fermented milk with Bifidobacterium infantis,Bifidobacterium bifidum, Bifidobacterium animalis, Lactobacillusacidophilus, or Lactobacillus paracasei on the growth of breast tumourcells MCF7 have demonstrated that the various fermented milks arecapable, even if to varying degrees, of inhibiting the growth of tumourcells. The anti-proliferative effect cannot be correlated to thepresence of the bacteria in the fermented milk, to the milk or to thefractions thereof; the hypothesis is for the presence of a solublecompound produced ex novo from the lactic bacteria during fermentationof the milk or microbial transformation of some components of the milkinto a biologically active form (Biffi. A. et al Nutr. Cancer 28:93,1997).

Many micro-organisms use arginine as a source of carbon, nitrogen andenergy. Arginine deiminase transforms arginine in the presence of waterinto citrulline and ammonia. That enzymatic procedure has beenencountered in a variety of pathogenic or potentially pathogenic isbacteria such as Pseudomonas sp and Bacillus sp. and in some types ofmycoplasms. It has been demonstrated that this system plays a part inoral ecology, in protecting less acid-tolerant organisms during the fallin the pH to 4, or even lower values, in dental plaque, duringglycolysis caused by bacteria which are more resistant to acidity(Curran. T. M. Appl. Environ. Microbio. 61:4494, 1995).

Studies have been conducted on arginine deiminase which can be obtainedfrom mycoplasms, to be used as a cure for cancer (Takaku, H. et al Jpn.J. Cancer Res. 1:840, 1995). Mycoplasms are micro-organisms which aresimilar to bacteria which, unlike the latter, lack a cell wall and thegenome of which is around ⅙ of that of E. coli; however they can bepathogenic for man, animals and for plants and in addition they cannotbe easily handled due to the absence of a cell wall. Purification wasthus implemented in respect of the enzyme arginine deiminase which canbe obtained from mycoplasms, which behaves like an immunogen and whichis not free from undesired effects if used in vivo (McGarrity, J. G. etal U.S. Pat. No. 5,372,942). Other micro-organisms endowed with argininedeiminase (such as for example Pseudomonas sp and Bacillus sp) cannot beused by virtue of their potential pathogenicity and pyrogenicity.

We have now surprisingly found that some bacteria are rich in argininedeiminase, in particular some Gram-positive bacteria and someGram-negative bacteria, and also some strains of lactic bacteria, inparticular of the species Lactobacillus brevis or Lactobacillusfermentum, more particularly the strain of Lactobacillus brevis referredto as CD2 deposited with the DSM—Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, Brunswick, Germany, under the access number DSM11988, are capable of inducing apoptosis and can therefore be used forprevention or therapy in respect of clinical situations characterised byinsufficient or absent apoptosis or by inflammation.

The above-mentioned bacteria have surprisingly shown an argininedeiminase capable of inducing apoptosis and they can be used as such orafter suitable lyophilisation or also after sonication. Indeed inaccordance with the present invention the bacteria in question can belive or sonicated and the level of concentration can fluctuate from1×10¹ CFU to 1×10¹³ CFU per gram of composition, according to thedesired effect and the amount of arginine deiminase which they have. Thesame bacterial strains can be used to reduce or terminate aninflammatory reaction caused by nitric oxide (NO). NO which issynthesised from L-arginine by means of nitric oxide synthase (NOS) isan intra- and intercellular messenger with numerous biological actions.Alterations in the level of synthesis of NO are at the basis of numerousother physiopathological conditions such as arterial hypertension, renalinsufficiency, septic shock, vasodilation induced by hypoxia, vasospasmresulting from subarachnoid haemorrhage, neuronal destruction invascular infarction and other neurodegenerative conditions, chronicinflammatory pathologies, anaphylaxis and immunodeficiency. Argininedeiminase converts arginine into citrulline and NH₃ without theproduction of nitric oxide and can thus have an anti-inflammatory andcurative or remedial effect, for example in intestinal malabsorption andpancreatic insufficiency with modulation for example of the metabolicand/or nutritional state of the subject. An effect which is referred toby way of non-limiting example can be that of reducing the levels ofoxalates and/or phosphates in blood and urine.

Non-limiting examples of diseases or disorders which can be treatedand/or prevented by using bacteria which are rich in arginine deiminaseare tumours in general in particular colon-rectal cancer, cancer of theliver, gliomae, neuroblastomae, squamocellular oral carcinoma, lymphoidtumours, cancer of the prostate gland, cancer of the bladder, cancer ofthe breast, cancer of the pleura and the peritoneum, serious myasthenia,systemic lupus erythematosus, and other auto-immune diseases includingthose of the thyroid, diseases characterised by acute and/or chronicinflammatory processes, bronchial asthma, intestinal inflammatorydiseases, gastrites, duodenites, gastric ulcers, duodenal ulcers,pneumonias and pleurisies, infections from adenovirus, baculovirus andin general supported by a viral agent, diseases characterised by acuteand/or chronic inflammatory and/or degenerative processes of the centraland/or peripheral nervous system, pancreatites, endomyocardites andischaemic damage (myocardiac, retinal, cerebral and renal),urolithiases, nephrocalcinoses, hyperoxaluria, hyperphosphaturia,nephroalteration in the systemic and/or district arterial and/or venouspressure such as portal hypertension, vaginoses and vaginites,procto-haemerrhoidal inflammations, prostates, sinusites and otites,conjunctivites, gingivites, periodontopathy, anaphylactic phenomena andimmunodeficiencies.

Such micro-organisms which are rich in arginine deiminase can be usedindividually or in combination with each or with other lactic bacteriasuch as Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacilluscasei, Lactobacillus catenaforme, Lactobacillus cellobiosus,Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillusdelbrueckii, Lactobacillus lensenii, Lactobacillus leichmanii,Lactobacillus minutus, Lactobacillus plantarum, Lactobacillus rogosae,Lactobacillus salivarius, Bifidobacterium adolescentis, Bifidobacteriumangulatum, Bifidobacterium bifidum, Bifidobacterium breve,Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacteriumeriksonii, Bifidobacterium infantis, Bifidobacterium longum,Bifidobacterium plantarum, Bifidobacterium pseudo-catenulatum,Bifidobacterium pseudolongum, Streptococcus lactis, Streptococcusraffinolactis, Streptococcus thermophilus, Acidaminococcus fermenta,Cytophaga fermentans, Rhodoferax fermentans, Cellulomonas fermentans andZymomonas mobilis.

Still in accordance with the present invention the bacteria can be usedin association with arginine deiminase, sphingomyelinase or otherenzymes, with cortisone, anti-inflammatory, immuno-modulant, cytostatic,immunological, endocrinological, vascular, anaesthetic, and vasodilatorydrugs, growth factors, cytokines, ceramides, vitamins and minerals,lipids, amino acids and carbohydrates, formulations for enteric use anddietetic, prebiotic or probiotic supplements, and with excipientscommonly used in the pharmaceutical industry or in thefoodstuffs/dietetic field. The preferred form of administration isorally but it is not limitative in relation to possible topical, rectal,nasal or parenteral administration. The composition of the inventionwill thus be in the form of pills or tablets, capsules, globuli,suppositories, emulsions, suspensions, stick-on plasters, creams,ointments, sprays, collyria, collutoria or dentifrices.

The following examples which are set forth by way of non-limitingillustration will illustrate the present invention in greater detail.

EXAMPLE 1 Induction of Apoptosis in Various Cellular Systems

Cells used:

1. Normal:

PBL (human peripheral blood lymphocytes)

HS27 (normal human fibroblasts)

HaCaT (eternalised normal human keratinocytes)

2. Tumoral:

Jurkat (human T leukaemia)

P815 (murine mastocytoma)

J744 (murine tumoral macrophages)

The cells were cultivated in suitable medium with serum (10%) at 37° C.(5%CO₂) for 18-72 hours in the presence or absence of sonicatedpreparations in buffered solution of phosphates (PBS) of L. brevis(final concentration in the cellular suspensions: 100 mg/10 ml). At theend of the incubation operation the cells were counted and vitalitydetermined on the basis of exclusion of the dye tripan blue. Possibleinduction of death by apoptosis in the cells treated with the bacteriawas determined on the basis of:

morphology under an optical microscope after colouring withhaematoxylin/eosin,

colouring with acridine orange/ethidium bromide detected by means offluorescence microscopy and cytofluorimetry, and

detection of DNA laddering by means of agarose gel electrophoresis ofthe DNA.

TABLE I Apoptosis (%) Controls L. brevis (CD2) PBL 0 0 HS27 0 0 HaCaT 00 Jurkat 2-3 15-20 P815 0 1-3 J744 0 1-3

The above-reported results indicate clearly that treatment for 14-18hours with sonicated bacteria of the invention determines the inductionof significant levels of apoptosis in tumoral cells, while not givingrise to any effect on the normal cell systems analysed.

Example 2 Demonstration of the Presence of Arginine Deiminase inBacterial Strains

The activity of arginine deiminase in some bacterial strains wasdetermined on the basis of conversion in an aqueous solution ofradio-marked arginine into citrulline and NH₃. The presence of suitableinhibitors (L-N-nitro-arginine methyl ester HCl and L-valine) capable ofspecifically inhibiting other enzymes which effect conversion of thearginine (nitric oxide synthase and arginase respectively) made itpossible to attribute the enzymatic activity determined to the argininedeiminase and not to other enzymes. In addition the use of a specificinhibitor in respect of arginine deiminase (formamidine) made itpossible to confirm the soundness of the results.

TABLE 2 Activity of arginine deiminase (expressed as pmol of radioactivecitrulline produced/mg bacterial proteins per minute Bacterial strainpmol citrulline/mg proteins/min L. brevis CD2 6.72 L. fermentum 0.46 L.casei 0.13 L. acidophilus 0.002 L. plantarum 0.05 B. bifidum 0.03 S.thermophilus 0.020

Bacterial strains are considered as useful for the purposes of thepresent invention, which have values of greater than 0.1 pmolcitrulline/mg bacterial proteins/min.

The presence of inhibitors of nitric oxide synthase (L-NAME,L-nitromonomethyl arginine) or arginase (L-valine) did not in any wayinfluence the enzymatic activity in regard to conversion of arginine tocitrulline, thus making it possible to attribute the generation ofcitrulline observed with the various bacteria to the arginine deiminase.In addition the absence in the analysis system of calcium and calmodulinwhich are indispensable in terms of the activity of the constituentnitric oxide synthase did not in any way modify the activity in terms ofconversion of the arginine on the part of bacteria, further confirmingthat the enzyme responsible for the latter is arginine deiminase.

The results set out hereinafter demonstrate that the activity ofarginine deiminase encountered in the bacteria in question was furthercapable of completely inhibiting both the activity of constituent nitricoxide synthase (NOS) and that of inducible NOS, probably because thepresence thereof involves deprivation of the substrate (arginine) of thevarious forms of nitric oxide synthase. For that purpose rat cerebellumextracts and rat peritoneal macrophages stimulated in vitro withlipopolysaccharide of E. coli (100 mg/ml) and interferon (100 U/ml)respectively were used as positive controls for constituent NOS and forinducible NOS.

TABLE 3 Activity of constituent and inducible nitric oxide synthase andarginine deiminase Citrulline (pmol/5 Sample microliters) Cerebellum0.24 Cerebellum + L-NAME 0.01 Cerebellum + calcium chelating agent(EGTA) 0.01 Cerebellum more inhibitor of calmodulin (W13) 0.01Cerebellum + L. brevis CD2 (5 micrograms) 0.72 Cerebellum + L. brevisCD2 + L-NAME 0.8 Cerebellum + L. brevis CD2 + EGTA 0.74 Cerebellum + L.brevis CD2 + W13 0.76 Cerebellum + L. fermentum (30 microgrammes) 0.38Cerebellum + L. fermentum + L-NAME 0.4 Cerebellum + L. fermentum + EGTA0.4 Cerebellum + L. fermentum + W13 0.39 Untreated macrophages 0Macrophages + LPS + IFN 0.32 Macrophages + LPS + IFN + L-NAME 0Macrophages + L. brevis CD2 (5 micrograms) 0.76 Macrophages + L. brevisCD2 + L-NAME 0.78 Macrophages + LPS + IFN + L. brevis CD2 0.81Macrophages + LPS + IFN + L. brevis CD2 + L-NAME 0.82 Macrophages + L.fermentum (30 micrograms) 0.4 Macrophages + L. fermentum + L-NAME 0.41Macrophages + LPS + IFN + L. fermentum 0.42 Macrophages + LPS + IFN + L.fermentum + L-NAME 0.4

It seems to be evident that not all the bacteria have an enzymaticactivity in respect of arginine deiminase of a significant level for thepurposes of the present invention (Table 2) and that the strains whichare endowed therewith inhibit both constituent NOS and inducible NOS, asis confirmed by the persistent presence of high values of citrullineeven in the presence of specific inhibitors of the two types of NOS(Table 3).

Example 3

4 patients were treated, suffering from pouchitis, a non-specificinflammation of the ileal reservoir, which is most frequentlycomplicated in the long term by the occurrence of ileo-ano-anastomosisfor ulcerative colitis. It has recently been suggested that pouchitis isthe result of inflammatory NO-mediated damage. The subjects, allvolunteers, were treated for 2 months with a lyophilised preparation ofL. brevis CD2 at a concentration of 5×10¹⁰ CFU/gr, by mouth, at a dosageof 6 g/day. Before and after the treatment a biopsy sample was takenfrom the mucous membrane of the pouch, which was subjected tohomogenisation and then to dosage of the citrulline by means of analysisof the conversion of radio-marked arginine into citrulline.

TABLE 4 Effect of the treatment with CD2 on the activity of induciblenitric oxide synthase in intestinal biopsies of patients with pouchitisCitrulline (pmol/mg proteins/min) Patients T0 T1 1 2.95 0.89 2 1.15 0.563 0.56 0.5 4 0.47 0.28 5 0.7 0.5

The treatment with CD2 afforded a significant reduction in the levels ofactivity of inducible nitric oxide synthase.

What is claimed is:
 1. A biologically pure strain of Lactobacillusbrevis CD2 deposited with the DSM-Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, Brunswick, Germany, under the access number DSM11988, having an activity of arginine deiminase expressed as picomolscitrulline/mg bacterial proteins/min of not less than 0.1 and theirdescendants, mutants and derivatives which possess said activity.
 2. Acomposition comprising the biologically pure strain of claim 1 togetherwith a diluent or a carrier.
 3. An enterally-administerablepharmaceutical composition which comprises the strain of claim
 1. 4. Thecomposition according to claim 3 wherein the strain is lyophilised orsonicated.
 5. The composition according to claim 3, wherein theconcentration of the strain is from 1×10¹ CFU to 1×10¹³ CFU per gram ofcomposition.
 6. The composition according to claim 4, wherein theconcentration of the strain is from 1×10¹ CFU to 1×10¹³ CFU per gram ofcomposition.
 7. The composition according to claim 3, 4, 5 or 6, whereinsaid strain is administered individually or in association withLactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus casei,Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacilluscurvatus, Lactobacillus delbrueckii, Lactobacillus jensenii,Lactobacillus leichmanii, Lactobacillus minutus, Lactobacillusplantarum, Lactobacillus rogosae, Lactobacillus salivarius,Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacteriumbifidum, Bifidobacterium breve, Bifidobacterium catenulatum,Bifidobacterium dentium, Bifidobacterium eriksonii, Bifidobacteriuminfantis, Bifidobacterium longum, Bifidobacterium plantarum,Bifidobacterium pseudo-catenulatum, Bifidobacterium pseudolongum,Bifidobacterium raffinolactis, Streptococcus termophilus,Acidaminococcus fermenta, Cytophaga fermentans, Rhodoferax fermentans,Cellulomonas fermentans and Zymomonas mobilis or in association witharginine deiminase, sphingomyelinase or other enzymes, with cortisone,anti-inflammatory, immuno-modulant, cytostatic, immunological,endocrinological, vascular, anaesthetic, and vasodilatory drugs, growthfactors, cytokines, ceramides, vitamins and minerals, lipids, aminoacids and carbohydrates, formulations for enteric use and dietetic,prebiotic or probiotic supplements, and with pharmacologically ordietetically excipients.